Use of EZ::TN™ Transposomes™ for Genetic Analysis and Direct Sequencing of Bacterial Genomic DNA

The genomes of over sixty bacterial species are sequenced or are currently undergoing sequencing. Sequencing tools have now progressed to the point at which the chromosomal DNA of small bacterial genomes can be directly sequenced without molecular cloning.1 Although direct sequencing has not been attempted for large-scale sequencing projects, the ability to directly sequence bacterial DNA has applications in gap filling and characterization of mutations. In addition, as more bacterial genome data is collected there is a need for global techniques for analyzing the functions of genes. Transposons have long been recognized as powerful tools for inserting sequencing primer binding sites and for creating gene "knockouts" (insertional mutagenesis) in microorganisms.

However, traditional transposition systems are often difficult to work with and limited in the number of microbial species in which they can be used. EZ::TN Transposomes, based on the pioneering work of Reznikoff and Goryshin,2,3 offer a new, faster, simpler and more efficient method to randomly introduce transposons into the genomes of many different microorganisms. An EZ::TN Transposome is a stable synaptic complex formed between the hyperactive Tn5 transposase and a Tn5-derived transposon. Although not required, the transposons used typically contain a selectable marker (e.g. antibiotic resistance gene) which allows rapid selection of transposed cells. Although EZ::TN Transposomes are normally formed transiently during transposition, a stable Transposome can be formed and isolated in the absence of Mg2+

EZ::TN Transposomes are stable enough to be electroporated into living cells3,4 (Figure 2). Once inside the cell, the Transposome is activated, presumably by the Mg2+ within the host's cellular environment. Once activated, the EZ::TN Transposome efficiently and randomly inserts its transposon into the genomic DNA of the host cell. By screening antibiotic-selected transposition clones for a desired phenotype, gene knockouts in many open reading frames (ORFs) can be found. Thus, EZ::TN Transposomes eliminate the need for transductions or matings to mobilize transposons. In this report, we extend the use of EZ::TN Transposomes to randomly create insertion mutants in Salmonella typhimurium, Proteus vulgaris and Pseudomonas sp. Importantly, we also demonstrate that EZ::TN Transposomes facilitate direct DNA sequencing of mutated bacterial genomic DNA.

Methods

Transposon mutagenesis

EZ::TN Tnp Transposomes were formed using purified, hyperactive EZ::TN Transposase and an EZ::TN Transposon containing a kanamycin-selectable marker. The standard reaction for EZ::TN Tnp Transposome formation is a mixture of 5 µl of 100 µg/ml Transposon in 10 mM Tris-HCl, pH 7.5; 1 mM EDTA, 10 µl of 1 U/µl EZ::TN Transposase and 5 µl of 100% glycerol. This procedure can be scaled up or scaled down as needed. EZ::TN Tnp Transposomes were formed by incubating the mix at 37°C for 10 minutes. One-microliter aliquots were used for electroporation. The remaining EZ::TN Tnp Transposome can be stored for up to 1 year at -20°C without loss of activity.

Cell electroporation

Proteus vulgaris was obtained from ATCC (number 13,315) and Pseudomonas sp. (MMSS-8 strain) was from the University of Wisconsin Bacteriology Department Stock Culture Collection. Electrocompetent cells were prepared in the same manner for all species. Cells were grown to mid-log phase at 37°C in LB broth with shaking, then chilled, harvested by centrifugation and washed with deionized water three times before suspending them in ice-cold 10% glycerol in deionized water. Cells were stored frozen at -70°C in 100-µl aliquots until used.

Prior to use, cells were thawed on ice. Fifty microliters were transferred to a 2.0-mm gap electroporation cuvette. One microliter of EZ::TN Tnp Transposome was added and the cells were electroporated at 2500 V and 5 microseconds time constant using an Eppendorf Multiporator. Transposition clones were selected by plating on LB plates containing 50 µg per ml of kanamycin for S. typhimurium and P. vulgaris or 300 µg per ml of kanamycin for Pseudomonas sp.

Bacterial DNA isolation

Individual Kanr transposition clones were grown overnight at 37°C in LB Broth containing 50 µg/ml kanamycin for S. typhimurium and P. vulgaris and 300 mg/ml kanamycin for Pseudomonas sp. Genomic DNA was purified using the MasterPure™ Complete DNA Purification Kit (Epicentre).

Direct genomic DNA sequencing and sequence analysis

Transposon insertion sites were sequenced bidirectionally using sequencing primers specific for the ends of the inserted transposon. Two to three micrograms of bacterial genomic DNA and 5-12 pmoles of primer were used in "2X" Big Dye Terminator sequencing reactions according to the manufacturer's protocols (PE Biosystems, Foster City CA). Bacterial genomic DNA does not require restriction endonuclease digestion or shearing to serve as a DNA sequencing template. Samples were cycled (DNA Engine, MJ Research, Waltham, MA) for 4 min at 95°C, then 60 cycles of 30 sec at 95°C and 4 min at 60°C followed by 4°C indefinitely. Sequencing reactions were purified by gel filtration with a Centri-Sep spin column (Princeton Separations, Princeton, NJ) , concentrated by ethanol precipitation, washed with 70% ethanol, and resuspended in 20 ml of Template Supression Reagent (PE Biosystems, Foster City, CA). After denaturing at 95°C for 5 min, the samples were injected into an ABI 310 Genetic Analyzer (PE Biosystems, Foster City, CA) and analyzed with ABI version 3.3 sequence analysis software. Transposon insertion sites can also be sequenced using radioactive and LI-COR sequencing methods (Ronald Meis, EPICENTRE, unpublished data). The genomic transposition sites were located using BLAST programs maintained at the NCBI web site of the National Library of Medicine.

The primer EB-L was used for sequencing 16S rDNA.5 Cycle sequencing and sample preparation and analysis were the same as above.